Analytical Performance and Inter-Method Agreement of a Laboratory-Developed CMV qPCR Assay in Clinical Plasma Samples

Area of Science:

• Virology
• Molecular Diagnostics
• Transplantation Medicine

Background:

• Cytomegalovirus (CMV) viral load monitoring is crucial for preemptive treatment in transplant patients.
• Evaluating new diagnostic methods is essential for improving patient care and outcomes.

Purpose of the Study:

• To assess the analytical performance of a novel laboratory-developed CMV real-time PCR (qPCR) assay.
• To determine the inter-method agreement between the new assay and a commercial reference method for CMV viral load quantification.

Main Methods:

• 100 EDTA plasma samples were analyzed using both the laboratory-developed CMV qPCR test and the Roche Cobas® CMV reference method.
• Analytical sensitivity was determined, and the limit of detection (LoD95) was calculated via probit regression.
• Spearman rank correlation and Bland-Altman analysis were employed to evaluate quantitative results and inter-method agreement.

Main Results:

• The laboratory-developed CMV qPCR test exhibited an LoD95 of 63.8 copies/µL.
• A weak, non-significant correlation (ρ = 0.32, p = 0.22) was found between the new assay and the reference method for quantitative results.
• Bland-Altman analysis indicated a mean difference of -0.48 log10 units, with most measurements within the 95% limits of agreement.

Conclusions:

• The developed CMV qPCR assay demonstrates acceptable analytical performance and inter-method agreement.
• Quantitative viral load monitoring using this assay, particularly at low CMV DNA concentrations, should be approached with caution.
• Further validation may be needed to optimize its clinical utility in transplant recipients.